黄瓜白粉病的PMA-qPCR方法建立及应用  

Establishment and application of a PMA-qPCR method for cucumber powdery mildew

作　　者：李金婷 张佐然 梁雅静 孔维文[1] 李云龙 何晓青[1] LI Jinting;ZHANG Zuoran;LIANG Yajing;KONG Weiwen;LI Yunlong;HE Xiaoqing(College of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083,China;Beijing Plant Protection Station,Beijing 100029,China)

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]北京市植物保护站,北京100029

出　　处：《福建农林大学学报（自然科学版）》2019年第5期550-555,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：中科院科技服务网络计划(KFJ-STS-ZDTP-036);国家自然科学基金(31700633、51108029);中央高校基本科研业务费专项(2017JC05);水利部公益性行业科研专项(201201032)

摘　　要：鉴于叠氮溴化丙锭(PMA)染料可辨别细菌活性,将其与实时荧光定量PCR(qPCR)相结合,建立PMA-qPCR方法,以检测黄瓜白粉病的活性致病菌.在黄瓜白粉菌核糖体DNA的ITS区设计引物,将扩增片段重组到pGM-Simple-T载体中构建标准质粒.以5×10-5×10^6个??μL^-1 6个梯度稀释质粒来绘制标准曲线,并做组内和组间重复.(1)qPCR标准曲线显示有良好的扩增结果,其相关系数(R2)为0.994.(2)利用qPCR检测臭氧处理后PMA染料处理组和对照组中有活性的黄瓜白粉菌数量.结果显示,PMA染料处理组拷贝数为4.335×10^2个??μL^-1,对照组拷贝数为9.448×10^6个??μL^-1.(3)利用PMA-qPCR方法检测臭氧处理后的黄瓜叶片不同部位(全组织、表面、内部)活菌数占总菌数的比例,处理50 min后的活菌比例分别较处理前下降了58.8%、67.0%、48.0%.这表明该方法可快速鉴定黄瓜白粉病中的活性致病菌数量,为该病的预防和控制提供技术支持.As an effective tool to diagnose bacterial viability,propidium monoazide(PMA)dye was combined with real-time fluo-rescent quantitative PCR(qPCR),named PMA-qPCR method,to detect active pathogenic bacteria in cucumber powdery mildew.Firstly,specific primers were designed in ITS conserved region of the ribosomal DNA of cucumber powdery mildew.Then the ampli-fied fragment was recombined into pGM-Simple-T vector to construct a standard plasmid,and followed by being diluted to concentra-tions of 5×10-5×10^6 copies??μL^-1 to draw a standard curve.The repeated experiments were conducted in intra-and inter-assays.(1)The standard curve of qPCR showed good repeatability with a linear correlation(R2)of 0.994.(2)The amounts of cucumber powdery mildew pathogen were quantified by qPCR,with the copy number being 4.335×10^2 copies ^μL^-1 for the PMA dye treat-ment groups and 9.448×10^6 copies·μL^-1 for the control.(3)Proportions of viable bacteria for whole leaf tissue,leaf epidermis,and mesophyll tissue decreased by 58.8%,67.0%and 48.0%respectively after ozone treatment for 50 min by PMA-qPCR method.Therefore,PMA-qPCR method proved to be a fast and accurate approach to identify the number of active pathogens in cucumber powdery mildew,and provide a technical support for the prevention and control of this disease.

关 键 词：黄瓜白粉病 PMA-qPCR 标准曲线 臭氧处理 

分 类 号：Q939.35[生物学—微生物学]